System-level optimization in spectroscopic photoacoustic imaging of prostate cancer.

This study presents a system-level optimization of spectroscopic photoacoustic (PA) imaging for prostate cancer (PCa) detection in three folds. First, we present a spectral unmixing model to segregate spectral system error (SSE). We constructed two noise models (NMs) for the laser spectrotemporal fluctuation and the ultrasound system noise. We used these NMs in linear spectral unmixing to denoise and to achieve high temporal resolution. Second, we employed a simulation-aided wavelength optimization to select the most effective subset of wavelengths. NMs again were considered so that selected wavelengths were not only robust to the collinearity of optical absorbance, but also to noise. Third, we quantified the effect of frame averaging on improving spectral unmixing accuracy through theoretical analysis and numerical validation. To validate the whole framework, we performed comprehensive studies in simulation and an in vivo experiment which evaluated prostate-specific membrane antigen (PSMA) expression in PCa on a mice model. Both simulation analysis and in vivo studies confirmed that the proposed framework significantly enhances image signal-to-noise ratio (SNR) and spectral unmixing accuracy. It enabled more sensitive and faster PCa detection. Moreover, the proposed framework can be generalized to other spectroscopic PA imaging studies for noise reduction, wavelength optimization, and higher temporal resolution.

An Evaluation of CXCR4 Targeting with PAMAM Dendrimer Conjugates for Oncologic Applications.

The chemokine receptor 4 (CXCR4) is a promising diagnostic and therapeutic target for the management of various cancers. CXCR4 has been utilized in immunotherapy, targeted drug delivery, and endoradiotherapy. Poly(amidoamine) [PAMAM] dendrimers are well-defined polymers with unique properties that have been used in the fabrication of nanomaterials for several biomedical applications. Here, we describe the formulation and pharmacokinetics of generation-5 CXCR4-targeted PAMAM (G5-X4) dendrimers. G5-X4 demonstrated an IC50 of 0.95 nM to CXCR4 against CXCL12-Red in CHO-SNAP-CXCR4 cells. Single-photon computed tomography/computed tomography imaging and biodistribution studies of 111In-labeled G5-X4 showed enhanced uptake in subcutaneous U87 glioblastoma tumors stably expressing CXCR4 with 8.2 ± 2.1, 8.4 ± 0.5, 11.5 ± 0.9, 10.4 ± 2.6, and 8.8 ± 0.5% injected dose per gram of tissue at 1, 3, 24, 48, and 120 h after injection, respectively. Specific accumulation of [111In]G5-X4 in CXCR4-positive tumors was inhibited by the peptidomimetic CXCR4 inhibitor, POL3026. Our results demonstrate that while CXCR4 targeting is beneficial for tumor accumulation at early time points, differences in tumor uptake are diminished over time as passive accumulation takes place. This study further confirms the applicability of PAMAM dendrimers for imaging and therapeutic applications. It also emphasizes careful consideration of image acquisition and/or treatment times when designing dendritic nanoplatforms for tumor targeting.

Pharmacodynamic measures within tumors expose differential activity of PD(L)-1 antibody therapeutics.

Macromolecules such as monoclonal antibodies (mAbs) are likely to experience poor tumor penetration because of their large size, and thus low drug exposure of target cells within a tumor could contribute to suboptimal responses. Given the challenge of inadequate quantitative tools to assess mAb activity within tumors, we hypothesized that measurement of accessible target levels in tumors could elucidate the pharmacologic activity of a mAb and could be used to compare the activity of different mAbs. Using positron emission tomography (PET), we measured the pharmacodynamics of immune checkpoint protein programmed-death ligand 1 (PD-L1) to evaluate pharmacologic effects of mAbs targeting PD-L1 and its receptor programmed cell death protein 1 (PD-1). For PD-L1 quantification, we first developed a small peptide-based fluorine-18-labeled PET imaging agent, [18F]DK222, which provided high-contrast images in preclinical models. We then quantified accessible PD-L1 levels in the tumor bed during treatment with anti-PD-1 and anti-PD-L1 mAbs. Applying mixed-effects models to these data, we found subtle differences in the pharmacodynamic effects of two anti-PD-1 mAbs (nivolumab and pembrolizumab). In contrast, we observed starkly divergent target engagement with anti-PD-L1 mAbs (atezolizumab, avelumab, and durvalumab) that were administered at equivalent doses, correlating with differential effects on tumor growth. Thus, we show that measuring PD-L1 pharmacodynamics informs mechanistic understanding of therapeutic mAbs targeting PD-L1 and PD-1. These findings demonstrate the value of quantifying target pharmacodynamics to elucidate the pharmacologic activity of mAbs, independent of mAb biophysical properties and inclusive of all physiological variables, which are highly heterogeneous within and across tumors and patients.

CRYßB2 enhances tumorigenesis through upregulation of nucleolin in triple negative breast cancer.

Expression of ß-crystallin B2 (CRYßB2) is elevated in African American (AA) breast tumors. The underlying mechanisms of CRYßB2-induced malignancy and the association of CRYßB2 protein expression with survival have not yet been described. Here, we report that the expression of CRYßB2 in breast cancer cells increases stemness, growth, and metastasis. Transcriptomics data revealed that CRYßB2 upregulates genes that are functionally associated with unfolded protein response, oxidative phosphorylation, and DNA repair, while down-regulating genes related to apoptosis. CRYßB2 in tumors promotes de-differentiation, an increase in mesenchymal markers and cancer-associated fibroblasts, and enlargement of nucleoli. Proteome microarrays identified a direct interaction between CRYßB2 and the nucleolar protein, nucleolin. CRYßB2 induces nucleolin, leading to the activation of AKT and EGFR signaling. CRISPR studies revealed a dependency on nucleolin for the pro-tumorigenic effects of CRYßB2. Triple-negative breast cancer (TNBC) xenografts with upregulated CRYßB2 are distinctively sensitive to the nucleolin aptamer, AS-1411. Lastly, in AA patients, higher levels of nucleolar CRYßB2 in primary TNBC correlates with decreased survival. In summary, CRYßB2 is upregulated in breast tumors of AA patients and induces oncogenic alterations consistent with an aggressive cancer phenotype. CRYßB2 increases sensitivity to nucleolin inhibitors and may promote breast cancer disparity.

Dual contrast agents for fluorescence and photoacoustic imaging: evaluation in a murine model of prostate cancer.

Prostate-specific membrane antigen (PSMA) is a promising diagnostic and therapeutic target for prostate cancer (PC). Poly(amidoamine) [PAMAM] dendrimers serve as versatile scaffolds for imaging agents and drug delivery that can be tailored to different sizes and compositions depending upon the application. We have developed PSMA-targeted PAMAM dendrimers for real-time detection of PC using fluorescence (FL) and photoacoustic (PA) imaging. A generation-4, ethylenediamine core, amine-terminated dendrimer was consecutively conjugated with on average 10 lysine-glutamate-urea PSMA targeting moieties and a different number of sulfo-cyanine7.5 (Cy7.5) near-infrared dyes (2, 4, 6 and 8 denoted as conjugates II, III, IV and V, respectively). The remaining terminal primary amines were capped with butane-1,2-diol functionalities. We also prepared a conjugate composed of Cy7.5-lysine-suberic acid-lysine glutamate-urea (I) and control dendrimer conjugate (VI). Among all conjugates, IV showed superior in vivo target specificity in male NOD-SCID mice bearing isogenic PSMA+ PC3 PIP and PSMA- PC3 flu xenografts and suitable physicochemical properties for FL and PA imaging. Such agents may prove useful in PC cancer detection and subsequent surgical guidance during excision of PSMA-expressing lesions.

Imaging of Fibroblast Activation Protein in Cancer Xenografts Using Novel (4-Quinolinoyl)-glycyl-2-cyanopyrrolidine-Based Small Molecules.

Fibroblast activation protein (FAP) has become a favored target for imaging and therapy of malignancy. We have synthesized and characterized two new (4-quinolinoyl)-glycyl-2-cyanopyrrolidine-based small molecules for imaging of FAP, QCP01 and [111In]QCP02, using optical and single-photon computed tomography/CT, respectively. Binding of imaging agents to FAP was assessed in six human cancer cell lines of different cancer types: glioblastoma (U87), melanoma (SKMEL24), prostate (PC3), NSCLC (NCIH2228), colorectal carcinoma (HCT116), and lung squamous cell carcinoma (NCIH226). Mouse xenograft models were developed with FAP-positive U87 and FAP-negative PC3 cells to test pharmacokinetics and binding specificity in vivo. QCP01 and [111In]QCP02 demonstrated nanomolar inhibition of FAP at Ki values of 1.26 and 16.20 nM, respectively. Both were selective for FAP over DPP-IV, a related serine protease. Both enabled imaging of FAP-expressing tumors specifically in vivo. [111In]QCP02 showed high uptake at 18.2 percent injected dose per gram in the U87 tumor at 30 min post-administration.

Twist activates miR-22 to suppress estrogen receptor alpha in breast cancer.

TWIST1 (Twist) is a basic helix-loop-helix transcription factor that is overexpressed in many cancers and promotes tumor cell invasion, metastasis, and recurrence. In this study, we demonstrate that Twist upregulates expression of microRNA 22 (miR-22) which, in turn, downregulates estrogen receptor alpha (ER) expression in breast cancer. Initial analysis of miR-22 and Twist expression in a panel of breast cancer cell lines showed a direct correlation between Twist and miR-22 levels with miR-22 being highly expressed in ER negative cell lines. Overexpressing Twist caused increased miR-22 levels while downregulating it led to decreased miR-22 expression. To characterize the upstream promoter region of miR-22, we utilized rapid amplification of cDNA ends and identified the transcription start site and the putative promoter region of miR-22. Mechanistically, we determined that Twist, in combination with HDAC1 and DNMT3B, transcriptionally upregulates miR-22 expression by binding to E-boxes in the proximal miR-22 promoter. We also established that miR-22 causes an increase in growth in 3D but not 2D cultures. Importantly, we observed a direct correlation between increased breast cancer grade and Twist and miR-22 expression. We also identified two potential miR-22 binding sites in the 3'-UTR region of ER and confirmed by promoter assays that miR-22 regulates ER expression by binding to both target sites. These results reveal a novel pathway of ER suppression by Twist through miR-22 activation that could potentially promote the ER negative phenotype in breast cancers.

Process validation, current good manufacturing practice production, dosimetry, and toxicity studies of the carbonic anhydrase IX imaging agent [111 In]In-XYIMSR-01 for phase I regulatory approval.

[111 In]In-XYIMSR-01 is a promising single-photon emission computed tomography (SPECT) imaging agent for identification of tumors that overexpress carbonic anhydrase IX. To translate [111 In]In-XYIMSR-01 to phase I trials, we performed animal toxicity and dosimetry studies, determined the maximum dose for human use, and completed the chemistry, manufacturing, and controls component of a standard regulatory application. The production process, quality control testing, stability studies, and specifications for sterile drug product release were based on United States Pharmacopeia chapters <823> and <825>, FDA 21 CFR Part 212. Toxicity was evaluated by using nonradioactive [113/115 In]In-XYIMSR-01 according to 21 CFR Part 58 guidelines. Organ Level INternal Dose Assessment/EXponential Modeling (OLINDA/EXM) was used to calculate the maximum single dose for human studies. Three process validation runs at starting radioactivities of ~800 MBq were completed with a minimum concentration of 407 MBq/ml and radiochemical purity of ≥99% at the end of synthesis. A single intravenous dose of 55 µg/ml of [113/115 In]In-XYIMSR-01 was well tolerated in male and female Sprague-Dawley rats. The calculated maximum single dose for human injection from dosimetry studies was 390.35 MBq of [111 In]In-XYIMSR-01. We have completed toxicity and dosimetry studies as well as validated a manufacturing process to test [111 In]In-XYIMSR-01 in a phase I clinical trial.

Prostate-specific membrane antigen (PSMA)-targeted photodynamic therapy enhances the delivery of PSMA-targeted magnetic nanoparticles to PSMA-expressing prostate tumors.

Enhanced vascular permeability in tumors plays an essential role in nanoparticle delivery. Prostate-specific membrane antigen (PSMA) is overexpressed on the epithelium of aggressive prostate cancers (PCs). Here, we evaluated the feasibility of increasing the delivery of PSMA-targeted magnetic nanoparticles (MNPs) to tumors by enhancing vascular permeability in PSMA(+) PC tumors with PSMA-targeted photodynamic therapy (PDT). Method: PSMA(+) PC3 PIP tumor-bearing mice were given a low-molecular-weight PSMA-targeted photosensitizer and treated with fluorescence image-guided PDT, 4 h after. The mice were then given a PSMA-targeted MNP immediately after PDT and monitored with fluorescence imaging and T2-weighted magnetic resonance imaging (T2-W MRI) 18 h, 42 h, and 66 h after MNP administration. Untreated PSMA(+) PC3 PIP tumor-bearing mice were used as negative controls. Results: An 8-fold increase in the delivery of the PSMA-targeted MNPs was detected using T2-W MRI in the pretreated tumors 42 h after PDT, compared to untreated tumors. Additionally, T2-W MRIs revealed enhanced peripheral intra-tumoral delivery of the PSMA-targeted MNPs. That finding is in keeping with two-photon microscopy, which revealed higher vascular densities at the tumor periphery. Conclusion: These results suggest that PSMA-targeted PDT enhances the delivery of PSMA-targeted MNPs to PSMA(+) tumors by enhancing the vascular permeability of the tumors.

Preclinical Evaluation of 213Bi- and 225Ac-Labeled Low-Molecular-Weight Compounds for Radiopharmaceutical Therapy of Prostate Cancer.

Prostate-specific membrane antigen (PSMA)-targeted radiopharmaceutical therapy is a new option for patients with advanced prostate cancer refractory to other treatments. Previously, we synthesized a ß-particle-emitting low-molecular-weight compound, 177Lu-L1 which demonstrated reduced off-target effects in a xenograft model of prostate cancer. Here, we leveraged that scaffold to synthesize α-particle-emitting analogs of L1, 213Bi-L1 and 225Ac-L1, to evaluate their safety and cell kill effect in PSMA-positive (+) xenograft models. Methods: The radiochemical synthesis, cell uptake, cell kill, and biodistribution of 213Bi-L1 and 225Ac-L1 were evaluated. The efficacy of 225Ac-L1 was determined in human PSMA+ subcutaneous and micrometastatic models. Subacute toxicity at 8 wk and chronic toxicity at 1 y after administration were evaluated for 225Ac-L1. The absorbed radiation dose of 225Ac-L1 was determined using the biodistribution data and α-camera imaging. Results:213Bi- and 225Ac-L1 demonstrated specific cell uptake and cell kill in PSMA+ cells. The biodistribution of 213Bi-L1 and 225Ac-L1 revealed specific uptake of radioactivity within PSMA+ lesions. Treatment studies of 225Ac-L1 demonstrated activity-dependent, specific inhibition of tumor growth in the PSMA+ flank tumor model. 225Ac-L1 also showed an increased survival benefit in the micrometastatic model compared with 177Lu-L1. Activity-escalated acute and chronic toxicity studies of 225Ac-L1 revealed off-target radiotoxicity, mainly in kidneys and liver. The estimated maximum tolerated activity was about 1 MBq/kg. α-Camera imaging of 225Ac-L1 revealed high renal cortical accumulation at 2 h followed by fast clearance at 24 h. Conclusion:225Ac-L1 demonstrated activity-dependent efficacy with minimal treatment-related organ radiotoxicity. 225Ac-L1 is a promising therapeutic for further clinical evaluation.

Enhancement of Radiotherapy with Human Mesenchymal Stem Cells Containing Gold Nanoparticles.

Radiotherapy is a common approach for the treatment of a wide variety of cancer types. Available data indicate that nanoparticles can enhance the effect of radiotherapy. We report the use of human mesenchymal stem cells to selectively deliver gold nanoparticles (GNPs) to MDA-MB-231 breast tumor xenografts in mice for the purpose of enhancing the effect of radiation therapy. Targeted delivery of GNPs to the tumor site, followed by irradiation of the tumor, enabled control of tumor growth. The results indicate that tumor-selective GNP delivery by human mesenchymal stem cells may represent a viable way to enhance the effectiveness of radiotherapy.

Engineered Fragments of the PSMA-Specific 5D3 Antibody and Their Functional Characterization.

Prostate-Specific Membrane Antigen (PSMA) is an established biomarker for the imaging and experimental therapy of prostate cancer (PCa), as it is strongly upregulated in high-grade primary, androgen-independent, and metastatic lesions. Here, we report on the development and functional characterization of recombinant single-chain Fv (scFv) and Fab fragments derived from the 5D3 PSMA-specific monoclonal antibody (mAb). These fragments were engineered, heterologously expressed in insect S2 cells, and purified to homogeneity with yields up to 20 mg/L. In vitro assays including ELISA, immunofluorescence and flow cytometry, revealed that the fragments retain the nanomolar affinity and single target specificity of the parent 5D3 antibody. Importantly, using a murine xenograft model of PCa, we verified the suitability of fluorescently labeled fragments for in vivo imaging of PSMA-positive tumors and compared their pharmacokinetics and tissue distribution to the parent mAb. Collectively, our data provide an experimental basis for the further development of 5D3 recombinant fragments for future clinical use.

Preclinical Evaluation of 203/212Pb-Labeled Low-Molecular-Weight Compounds for Targeted Radiopharmaceutical Therapy of Prostate Cancer.

Targeted radiopharmaceutical therapy (TRT) using α-particle radiation is a promising approach for treating both large and micrometastatic lesions. We developed prostate-specific membrane antigen (PSMA)-targeted low-molecular-weight agents for 212Pb-based TRT of patients with prostate cancer (PC) by evaluating the matching γ-emitting surrogate, 203Pb. Methods: Five rationally designed low-molecular-weight ligands (L1-L5) were synthesized using the lysine-urea-glutamate scaffold, and PSMA inhibition constants were determined. Tissue biodistribution and SPECT/CT imaging of 203Pb-L1-203Pb-L5 were performed on mice bearing PSMA(+) PC3 PIP and PSMA(-) PC3 flu flank xenografts. The absorbed radiation dose of the corresponding 212Pb-labeled analogs was determined using the biodistribution data. Antitumor efficacy of 212Pb-L2 was evaluated in PSMA(+) PC3 PIP and PSMA(-) PC3 flu tumor models and in the PSMA(+) luciferase-expressing micrometastatic model. 212Pb-L2 was also evaluated for dose-escalated, long-term toxicity. Results: All new ligands were obtained in high yield and purity. PSMA inhibitory activities ranged from 0.10 to 17 nM. 203Pb-L1-203Pb-L5 were synthesized in high radiochemical yield and specific activity. Whole-body clearance of 203Pb-L1-203Pb-L5 was fast. The absorbed dose coefficients (mGy/kBq) of the tumor and kidneys were highest for 203Pb-L5 (31.0, 15.2) and lowest for 203Pb-L2 (8.0, 4.2). The tumor-to-kidney absorbed dose ratio was higher for 203Pb-L3 (3.2) and 203Pb-L4 (3.6) than for the other agents, but with lower tumor-to-blood ratios. PSMA(+) tumor lesions were visualized through SPECT/CT as early as 0.5 h after injection. A proof-of-concept therapy study with a single administration of 212Pb-L2 demonstrated dose-dependent inhibition of tumor growth in the PSMA(+) flank tumor model. 212Pb-L2 also demonstrated an increased survival benefit in the micrometastatic model compared with 177Lu-PSMA-617. Long-term toxicity studies in healthy, immunocompetent CD-1 mice revealed kidney as the dose-limiting organ. Conclusion:203Pb-L1-203Pb-L5 demonstrated favorable pharmacokinetics for 212Pb-based TRT. The antitumor efficacy of 212Pb-L2 supports the corresponding 203Pb/212Pb theranostic pair for PSMA-based α-particle TRT in advanced PC.

A side-by-side evaluation of [18F]FDOPA enantiomers for non-invasive detection of neuroendocrine tumors by positron emission tomography.

Neuroendocrine tumors (NETs) are an extremely heterogenous group of malignancies with variable clinical behavior. Molecular imaging of patients with NETs allows for effective patient stratification and treatment guidance and is crucial in selection of targeted therapies. Positron emission tomography (PET) with the radiotracer L-[18F]FDOPA is progressively being utilized for non-invasive in vivo visualization of NETs and pancreatic ß-cell hyperplasia. While L-[18F]FDOPA-PET is a valuable tool for disease detection and management, it also exhibits significant diagnostic limitations owing to its inherent physiological uptake in off-target tissues. We hypothesized that the D-amino acid structural isomer of that clinical tracer, D-[18F]FDOPA, may exhibit superior clearance capabilities owing to a reduced in vivo enzymatic recognition and enzyme-mediated metabolism. Here, we report a side-by-side evaluation of D-[18F]FDOPA with its counterpart clinical tracer, L-[18F]FDOPA, for the non-invasive in vivo detection of NETs. In vitro evaluation in five NET cell lines, including invasive small intestinal neuroendocrine carcinomas (STC-1), insulinomas (TGP52 and TGP61), colorectal adenocarcinomas (COLO-320) and pheochromocytomas (PC12), generally indicated higher overall uptake levels of L-[18F]FDOPA, compared to D-[18F]FDOPA. While in vivo PET imaging and ex vivo biodistribution studies in PC12, STC-1 and COLO-320 mouse xenografts further supported our in vitro data, they also illustrated lower off-target retention and enhanced clearance of D-[18F]FDOPA from healthy tissues. Cumulatively our results indicate the potential diagnostic applications of D-[18F]FDOPA for malignancies where the utility of L-[18F]FDOPA-PET is limited by the physiological uptake of L-[18F]FDOPA, and suggest D-[18F]FDOPA as a viable PET imaging tracer for NETs.

177Lu-labeled low-molecular-weight agents for PSMA-targeted radiopharmaceutical therapy.

PURPOSE: To develop a prostate-specific membrane antigen (PSMA)-targeted radiotherapeutic for metastatic castration-resistant prostate cancer (mCRPC) with optimized efficacy and minimized toxicity employing the ß-particle radiation of 177Lu. METHODS: We synthesized 14 new PSMA-targeted, 177Lu-labeled radioligands (177Lu-L1-177Lu-L14) using different chelating agents and linkers. We evaluated them in vitro using human prostate cancer PSMA(+) PC3 PIP and PSMA(-) PC3 flu cells and in corresponding flank tumor models. Efficacy and toxicity after 8 weeks were evaluated at a single administration of 111 MBq for 177Lu-L1, 177Lu-L3, 177Lu-L5 and 177Lu-PSMA-617. Efficacy of 177Lu-L1 was further investigated using different doses, and long-term toxicity was determined in healthy immunocompetent mice. RESULTS: Radioligands were produced in high radiochemical yield and purity. Cell uptake and internalization indicated specific uptake only in PSMA(+) PC3 cells. 177Lu-L1, 177Lu-L3 and 177Lu-L5 demonstrated comparable uptake to 177Lu-PSMA-617 and 177Lu-PSMA-I&T in PSMA-expressing tumors up to 72 h post-injection. 177Lu-L1, 177Lu-L3 and 177Lu-L5 also demonstrated efficient tumor regression at 8 weeks. 177Lu-L1 enabled the highest survival rate. Necropsy studies of the treated group at 8 weeks revealed subacute damage to lacrimal glands and testes. No radiation nephropathy was observed 1 year post-treatment in healthy mice receiving 111 MBq of 177Lu-L1, most likely related to the fast renal clearance of this agent. CONCLUSIONS: 177Lu-L1 is a viable clinical candidate for radionuclide therapy of PSMA-expressing malignancies because of its high tumor-targeting ability and low off-target radiotoxic effects.

Development of [18F]FPy-WL12 as a PD-L1 Specific PET Imaging Peptide.

Expression of programmed cell death ligand 1 (PD-L1) within tumors is an important biomarker for guiding immune checkpoint therapies; however, immunohistochemistry-based methods of detection fail to provide a comprehensive picture of PD-L1 levels in an entire patient. To facilitate quantification of PD-L1 in the whole body, we developed a peptide-based, high-affinity PD-L1 imaging agent labeled with [18F]fluoride for positron emission tomography (PET) imaging. The parent peptide, WL12, and the nonradioactive analog of the radiotracer, 19FPy-WL12, inhibit PD-1/PD-L1 interaction at low nanomolar concentrations (half maximal inhibitory concentration [IC50], 26-32 nM). The radiotracer, [18F]FPy-WL12, was prepared by conjugating 2,3,5,6-tetrafluorophenyl 6-[18F]fluoronicotinate ([18F]FPy-TFP) to WL12 and assessed for specificity in vitro in 6 cancer cell lines with varying PD-L1 expression. The uptake of the radiotracer reflected the PD-L1 expression assessed by flow cytometry. Next, we performed the in vivo evaluation of [18F]FPy-WL12 in mice bearing cancer xenografts by PET imaging, ex vivo biodistribution, and blocking studies. In vivo data demonstrated a PD-L1-specific uptake of [18F]FPy-WL12 in tumors that is reduced in mice receiving a blocking dose. The majority of [18F]FPy-WL12 radioactivity was localized in the tumors, liver, and kidneys indicating the need for optimization of the labeling strategy to improve the in vivo pharmacokinetics of the radiotracer.

Evaluation of PSMA-Targeted PAMAM Dendrimer Nanoparticles in a Murine Model of Prostate Cancer.

The prostate-specific membrane antigen (PSMA) is a validated target for detection and management of prostate cancer (PC). It has also been utilized for targeted drug delivery through antibody-drug conjugates and polymeric micelles. Polyamidoamine (PAMAM) dendrimers are emerging as a versatile platform in a number of biomedical applications due to their unique physicochemical properties, including small size, large number of reactive terminal groups, bulky interior void volume, and biocompatibility. Here, we report the synthesis of generation 4 PSMA-targeted PAMAM dendrimers [G4(MP-KEU)] and evaluation of their targeting properties in vitro and in vivo using an experimental model of PC. A facile, one-pot synthesis gave nearly neutral nanoparticles with a narrow size distribution of 5 nm in diameter and a molecular weight of 27.3 kDa. They exhibited in vitro target specificity with a dissociation constant ( Kd) of 0.32 ± 0.23 µm and preferential accumulation in PSMA+ PC3 PIP tumors versus isogenic PSMA- PC3 flu tumors. Positron emission tomography-computed tomography imaging and ex vivo biodistribution studies of dendrimers radiolabeled with 64Cu, [64Cu]G4(MP-KEU), demonstrated high accumulation in PSMA+ PC3 PIP tumors at 24 h post-injection (45.83 ± 20.09% injected dose per gram of tissue, %ID/g), demonstrating a PSMA+ PC3 PIP/PSMA- PC3 flu ratio of 7.65 ± 3.35. Specific accumulation of G4(MP-KEU) and [64Cu]G4(MP-KEU) in PSMA+ PC3 PIP tumors was inhibited by the known small-molecule PSMA inhibitor, ZJ-43. On the contrary, G4(Ctrl), control dendrimers without PSMA-targeting moieties, showed comparable low accumulation of ∼1%ID/g in tumors irrespective of PSMA expression, further confirming PSMA+ tumor-specific uptake of G4(MP-KEU). These results suggest that G4(MP-KEU) may represent a suitable scaffold by which to target PSMA-expressing tissues with imaging and therapeutic agents.

MRI Assessment of Prostate-Specific Membrane Antigen (PSMA) Targeting by a PSMA-Targeted Magnetic Nanoparticle: Potential for Image-Guided Therapy.

Magnetic nanoparticle (MNP)-induced hyperthermia is currently being evaluated for localized prostate cancer. We evaluated the feasibility of tumor-selective delivery of prostate-specific membrane antigen (PSMA)-targeted MNPs in a murine model with high-resolution magnetic resonance imaging (MRI) after intravenous administration of MNPs at a concentration necessary for hyperthermia. A PSMA-targeted MNP was synthesized and evaluated using T2-weighted MRI, after intravenous administration of 50 mg/kg of the MNP. Significant contrast enhancement ( P < 0.0002, n = 5) was observed in PSMA(+) tumors compared to PSMA(-) tumors 24 h and 48 h after contrast agent administration. Mice were also imaged with near-infrared fluorescence imaging, to validate the MRI results. Two-photon microscopy revealed higher vascular density at the tumor periphery, which resulted in higher  peripheral accumulation of PSMA-targeted MNPs. These results suggest that the delivery of PSMA-targeted MNPs to PSMA(+) tumors is both actively targeted and passively mediated.

Evaluation of 111In-DOTA-5D3, a Surrogate SPECT Imaging Agent for Radioimmunotherapy of Prostate-Specific Membrane Antigen.

5D3 is a new high-affinity murine monoclonal antibody specific for prostate-specific membrane antigen (PSMA). PSMA is a target for the imaging and therapy of prostate cancer. 111In-labeled antibodies have been used as surrogates for 177Lu/90Y-labeled therapeutics. We characterized 111In-DOTA-5D3 by SPECT/CT imaging, tissue biodistribution studies, and dosimetry. Methods: Radiolabeling, stability, cell uptake, and internalization of 111In-DOTA-5D3 were performed by established techniques. Biodistribution and SPECT imaging were done on male nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice bearing human PSMA(+) PC3 PIP and PSMA(-) PC3 flu prostate cancer xenografts on the upper right and left flanks, respectively, at 2, 24, 48, 72, and 192 h after injection. Biodistribution was also evaluated in tumor-free, healthy male CD-1 mice. Blocking studies were performed by coinjection of a 10-fold and 50-fold excess of 5D3 followed by biodistribution at 24 h to determine PSMA binding specificity. The absorbed radiation doses were calculated on the basis of murine biodistribution data, which were translated to a human adult man using organ weights as implemented in OLINDA/EXM. Results:111In-DOTA-5D3 was synthesized with specific activity of approximately 2.24 ± 0.74 MBq/µg (60.54 ± 20 µCi/µg). Distribution of 111In-DOTA-5D3 in PSMA(+) PC3 PIP tumor peaked at 24 h after injection and remained high until 72 h. Uptake in normal tissues, including the blood, spleen, liver, heart, and lungs, was highest at 2 h after injection. Coinjection of 111In-DOTA-5D3 with a 10- and 50-fold excess of nonradiolabeled antibody significantly reduced PSMA(+) PC3 PIP tumor and salivary gland uptake at 24 h but did not reduce uptake in kidneys and lacrimal glands. Significant clearance of 111In-DOTA-5D3 from all organs occurred at 192 h. The highest radiation dose was received by the liver (0.5 mGy/MBq), followed by the spleen and kidneys. Absorbed radiation doses to the salivary and lacrimal glands and bone marrow were low. Conclusion:111In-DOTA-5D3 is a new radiolabeled antibody for imaging and a surrogate for therapy of malignant tissues expressing PSMA.

Peptide-based PET quantifies target engagement of PD-L1 therapeutics.

Immune checkpoint therapies have shown tremendous promise in cancer therapy. However, tools to assess their target engagement, and hence the ability to predict their efficacy, have been lacking. Here, we show that target engagement and tumor-residence kinetics of antibody therapeutics targeting programmed death ligand-1 (PD-L1) can be quantified noninvasively. In computational docking studies, we observed that PD-L1-targeted monoclonal antibodies (atezolizumab, avelumab, and durvalumab) and a high-affinity PD-L1-binding peptide, WL12, have common interaction sites on PD-L1. Using the peptide radiotracer [64Cu]WL12 in vivo, we employed positron emission tomography (PET) imaging and biodistribution studies in multiple xenograft models and demonstrated that variable PD-L1 expression and its saturation by atezolizumab, avelumab, and durvalumab can be quantified independently of biophysical properties and pharmacokinetics of antibodies. Next, we used [64Cu]WL12 to evaluate the impact of time and dose on the unoccupied fraction of tumor PD-L1 during treatment. These quantitative measures enabled, by mathematical modeling, prediction of antibody doses needed to achieve therapeutically effective occupancy (defined as >90%). Thus, we show that peptide-based PET is a promising tool for optimizing dose and therapeutic regimens employing PD-L1 checkpoint antibodies, and can be used for improving therapeutic efficacy.